It is often important for medical purposes to obtain nucleated fetal cells during pregnancy. For example, the procedures of amniocentesis or chrionic villae extraction are carried out for this purpose. But these methods are invasive, and carry a small but positive risk to the patient, and are complicated to perform. On the other hand, the purpose of my invention is to obtain nucleated fetal cells (fetal nucleated reticulocytes) from maternal blood, which eliminates the need for invasive methods. There are a small number of nucleated fetal red blood cells (reticulocytes) in maternal blood and my invention is a method and apparatus for separating these fetal cells from maternal blood.
Several methods have previously been proposed, involving fluorescent antibody antigen reactions (immunofluorescence), other antibody-antigen means, that depend on the difference between the cell walls of maternal and fetal blood cells, but all of these methods have been unsatisfactory because they have proved to be unreliable and expensive, and to involve some final additional technique to isolate the cells, such as flow cytometry. On the other hand, my invention does not use antibody-antigen means, but is based on the fundamental difference between maternal and fetal cells, namely that the former contains adult hemoglobin (HbA) while the latter contains fetal hemoglobin (HbF). The difference in the properties of these hemoglobin molecules themselves enables the fetal cells to be separated from the maternal cells by a magnetic field means.
Hemoglobin (Hb) is a tetrameric protein composed of four subchains, or globin monomers, each of which contains a heme group with an iron (Fe) atom at its center. The function of Hb is to carry oxygen (O.sub.2) to body tissues, and depends on the binding of O.sub.2 to the Fe in the heme group of each monomer. The iron is normally in the ferrous (Fe(II)) oxidation state whether or not the heme is oxygenated. The change from deoxygenated Hb (deoxyHb) to oxygenated Hb (oxyHb) involves a change in the electronic state of the Fe(II)-heme as indicated by the change in color of venous blood from the dark purplish hue to the brilliant red color of arterial blood. This change also explains why deoxyHb is paramagnetic, whereas oxyHb is non-paramagnetic.
My invention takes advantage of the main structural difference between nucleated adult and fetal red blood cells (rbc's), i.e., reticulocytes and erythrocytes, namely, the difference in the structure of the contained hemoglobin molecules. Maternal or adult hemoglobin (HbA) differs from fetal hemoglobin (HbF) in that the four subchains of HbA consist of two .alpha. chain monomers, and two .beta. chain monomers, whereas HbF is comprised of two .alpha. chain monomers and two .gamma. chain monomers. The result of this structural difference is that under normal physiologic conditions the O.sub.2 affinity of HbF is greater than that of HbA, enabling the fetal rbc's to obtain O.sub.2 from the maternal rbc's.
The affinity of Hb for O.sub.2 is given by the Hill equation for the chemical reaction [deoxyHb]+4[O.sub.2 ].revreaction.[oxyHb] with reaction rate constant K =[deoxyHb][O.sub.2 ].sup.4 /[oxyHb], giving the fractional yield Y, namely [oxyHb]/([deoxyHb]+[oxyHb]), as a function of [O.sub.2 ] where [O.sub.2 ] is the partial pressure of O.sub.2, usually denoted by pO.sub.2 : EQU Y=[pO.sub.2 ].sup.4 /(K+[pO.sub.2 ].sup.4 )
FIG. 1 shows the graph or Hill curve giving the yield of oxyHb as a function of the partial pressure of pO.sub.2 (i.e., the concentration, [.sub.2 ]).
If we graph the curves for the yield Y of both oxyHbA and oxyHbF as a function of pO.sub.2, as shown in FIG. 2, then it can be seen that at any given pO.sub.2, the yield of oxyHbF is greater than that of oxyHbA (i.e., see for instance, the vertical line).
Five physiologic factors can have an important influence on the curves, namely, the concentrations of CO.sub.2, H.sup.+, CI.sup.- and D-2,3-bisphosphoglycerate (BPG), and the temperature (T). FIG. 3 shows the effect of combinations of some of these factors. In general it can be said that decreasing the concentrations of CO.sub.2, H.sup.+, CI.sup.-, and BPG will move the curve to the left, and make the maximum slope of the curve somewhat steeper. Decreasing the temperature will also move the curve to the left. Decreases in the concentrations of adenosine triphosphate (ATP) and inositol hexaphosphate (IHP) will have a similar effect, although these factors are not as important as the five just discussed.
In summary: (a) deoxyHb is paramagnetic whereas oxyHb is not; (b) five main factors can influence the O.sub.2 affinity (or oxyHb yield curves), namely the concentrations of CO.sub.2, H.sup.+, CI.sup.-, and BPG, and the temperature T; and finally (c) HbF has a greater O.sub.2 affinity than HbA under the same pO.sub.2 condition. My invention makes use of these observations, combining them to produce a unique method and apparatus for separating fetal nucleated reticulocytes from maternal blood.
In particular, my invention takes advantage of the fact that, as can be seen from FIG. 2, at a particular value of pO.sub.2, the yield of oxyHbF can be significantly greater than that of oxyHbA. The in-vitro manipulation of the various factors can enhance this effect. This means that Hb in the nucleated fetal cells, i.e., HbF, will mostly be oxyHbF, whereas Hb in the nucleated maternal cells, i.e., HbA, will mostly be deoxyHbA. In an applied magnetic field, the deoxyHbA will be affected by the field, whereas the oxyHbF will not. Thus the maternal cells containing the deoxyHbA will be effected by the magnetic field, whereas the fetal cells containing the oxyHbF will not. The magnetic field can then effectuate the separation of nucleated maternal from nucleated fetal cells by acting on the respective HbA but not the HbF within the respective cells.
Despite extensive research on Hb and on methods of identifying and separating fetal nucleated reticulocytes from maternal blood, no one has heretofore combined the above described observations to carry out such an identification and separation.